Project Summary Tuberculosis (TB) causes more deaths worldwide than any other infectious disease, yet in the past, the impact of TB on children has been often dismissed or minimized. Poor diagnosis and reporting of tuberculosis (TB) in infants and children continue to hinder accurate estimates of the TB burden in children and the worldwide burden of tuberculosis remains high among children. The diagnosis of TB in children is challenging at present due to a lack of a sensitive point-of-care diagnostic tests, low access to and use of available tests, and difficulties of obtaining sputum specimens from children with TB. Moreover, available clinical tests need sufficient infrastructure support for the testing process such as a stable power supply, trained personnel, or lab facilities which preclude their use in peripheral settings. Many are also cost prohibitive for low resource countries where TB load is high. Currently, there is a great need for low cost rapid non-sputum TB diagnostics for children and a more accessible sample that can be obtained with minimal skill at primary health care level for use in an alternative diagnostic would be highly desirable for the diagnosis of TB in children. When children cough, they usually swallow the sputum. It has been shown that feces, the ultimate destination of swallowed sputum, is potentially a useful specimen for detection of TB in children. Indeed, viable Mycobacterium tuberculosis (Mtb) has been shown to be present in fecal samples using culture studies. More recently, there has been a number of attempts to adapt the GeneXpert test to feces from children. While the analysis of stool specimens by GeneXpert potentially represents a new diagnostic test for pediatric TB, its use in peripheral settings is severely limited. To solve this problem, we have designed and tested a rapid, low cost sputum-based diagnostic (REFtb) for peripheral settings that could be modified for use as a pediatric fecal test. The broad objective of the proposed research in this application is to adapt this sputum-based test to a fecal-based pediatric test. Based on preliminary data, we believe that with minimal optimization the sputum REFtb assay can easily be adapted from sputum to fecal matter as a diagnostic test. These studies will focus on adapting the sputum-based regents for use in a pediatric fecal-based assay and testing the fecal-based assay in pediatric fecal material. We plan to examine nonTB fecal material from children for interference activities to the REFtb test and test known inhibitors of the sputum-based REFtb test in order to develop an optimized reagent mix for the fecal-based test. Longer term we plan to continue development to develop a shelf-stable pediatric test and test the shelf-stable diagnostic in preliminary clinical trials. Completion of this proposed research will result in the development of a fecal-based pediatric TB diagnostic suitable for low resource peripheral settings. This fecal-based pediatric REFtb system overcomes the financial and geographical barriers that currently limit early TB detection so children with TB that currently go undiagnosed can now be diagnosed in a low-cost and rapid manner.